摘要
目的基于超高效液相色谱-四级杆静电场轨道阱质谱(UPLC-Q-Exactive MS/MS)研究D-半乳糖诱导衰老小鼠肾脏衰老的代谢轨迹,以及黄精多糖对D-半乳糖诱导衰老小鼠肾脏衰老的影响。方法将36只C57BL/6J小鼠随机分为6组:对照组(CON)、模型组(MOD)、黄精多糖低剂量组(PSP-L)、中剂量组(PSP-M)、高剂量组(PSP-H)和阳性药物组(VC)。为建立衰老模型,除对照组外,其他各组小鼠均往腹腔注射D-半乳糖。模型建立后,给予相应的中药[PSP,PSP-L:150 mg/(kg·d),PSP-M:300 mg/(kg·d),PSP-H:600 mg/(kg·d)]或阳性药物[抗坏血酸,300 mg/(kg·d)]进行干预。测定各组小鼠尿液和血清中肾功能的关键指标,如肌酐(Crea)、尿素氮(BUN)和尿酸(UA)水平,以及血清和肾脏中氧化应激的关键指标,包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px),以验证肾脏衰老模型的成功建立,并评价黄精多糖的影响。苏木精-伊红(HE)染色、过碘酸雪夫(PAS)染色和β-半乳糖苷酶染色用于评估肾脏病理变化。采用UPLC-Q-Exactive MS/MS分析了对照组、模型组和黄精多糖高剂量组血清、肾脏和尿液样本的代谢谱,并利用模式识别方法研究肾脏衰老的代谢轨迹和特征代谢物的鉴定。结果与年龄有关的小鼠肾组织病理学改变和肾功能受损与氧化应激指标有关。给予黄精多糖[PSP-H:600 mg/(kg·d)]后,与衰老相关的病理指标被调整到正常水平,衰老小鼠的肾功能和氧化应激得到改善,并显著改善了肾脏病理损伤。同时,通过UPLC-Q-Exactive MS/MS分析鉴定了潜在的生物标志物,并进一步以P<0.05为临界值分析形成了相关的代谢通路。结果表明,嘌呤代谢、鞘脂代谢、甘油磷脂代谢、色氨酸代谢和核黄素代谢是衰老相关的主要代谢通路。给予黄精多糖后,这些病理指标恢复到正常水平,与衰老过程相关的生物标志物,如单磷酸腺苷(AMP)、色氨酸和5-羟色氨酸,也在一定程度上表现出反向调节(促进合成)作用。结论基于UPLC-Q-Exactive MS/MS和多元统计分析的代谢组学方法可用于建立衰老小鼠的代谢谱。黄精多糖可通过干扰肾脏中的嘌呤代谢,鞘脂代谢,甘油磷脂代谢,色氨酸代谢和核黄素代谢,显示出对肾脏衰老的保护作用。
Objective To investigate the metabolic trajectory of kidney aging and the effects of Polygonatum sibiricum polysaccharides(PSP)against kidney aging in D-galactose(D-gal)-induced aging mice,based on ultra-performance liquid chromatography/Q-Exactive Orbitrap mass spectrometry(UPLC-Q-Exactive MS/MS).Methods A total of 36 C57 BL/6J mice were randomly allocated to six groups:control(CON),model(MOD),PSP low-dose(PSP-L),PSP medium-dose(PSP-M),PSP high-dose(PSP-H),and positive drug ascorbic acid(VC)groups.To create models of aging mice,D-gal was intraperitoneally administered to all other groups of mice except the CON group.After modeling,the appropriate Chinese medicine[PSP-L:150 mg/(kg·d),PSP-M:300 mg/(kg·d),PSP-H:600 mg/(kg·d)]or positive drug[ascorbic acid,300 mg/(kg·d)]was administered for intervention.Key markers of renal function in urine and serum of mice in each group,such as creatinine(Crea),urea nitrogen(BUN),and uric acid(UA)levels,as well as key indicators of oxidative stress in serum and kidney,including superoxide dismutase(SOD),catalase(CAT),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)were determined to validate the successful establishment of kidney aging models and to estimate the effects of PSP.Hematoxylin and eosin(HE),periodic acid Schiff(PAS),andβ-galactosidase staining were used to assess the renal pathological changes.The metabolic profiles of serum,kidney,and urine samples from CON,MOD,and PSP-H groups were analyzed by UPLC-Q-Exactive MS/MS,and pattern recognition methods were used to outline the metabolic trajectory of kidney aging and to identify the characteristic metabolites.Results Age-related alterations in renal histopathology and impaired renal function in mice were also associated with oxidative stress indicators.Following the injection of PSP[PSP-H:600 mg/(kg·d)],the pathological indices associated with aging were adjusted to normal levels,renal function and oxidative stress were improved in aging mice,and renal pathological damage was markedly improved.Meanwhile,the potential biomarkers were identified by UPLCQ-Exactive MS/MS analysis and were further analyzed to form related metabolic pathways,with P<0.05 as a threshold.The results showed that purine,sphingolipid,glycerophospholipid,tryptophan,and riboflavin metabolisms were the main metabolic pathways associated with aging.After administration of PSP,these pathological indices returned to normal levels,and biomarkers related to the aging process,such as adenosine monophosphate(AMP),tryptophan,and 5-hydroxytryptophan,also demonstrated,to some degree,reverse regulation(promoting synthesis).Conclusion Metabolomics methods based on UPLC-Q-Exactive MS/MS and multivariate statistical analysis can be adopted to establish metabolic profiles in aging mice.PSP has been shown to protect against kidney aging by interfering with the purine,sphingolipid,glycerophospholipid,tryptophan,and riboflavin metabolisms in the kidney.
作者
赵荻
Sharmeen Fayyaz
易子漾
刘昭
王艳
蔡萍
贺炜
ZHAO Di;Sharmeen Fayyaz;YI Ziyang;LIU Zhao;WANG Yan;CAI Ping;HE Wei(Graduate School,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;Husein Ebrahim Jamal Research Institute of Chemistry,International Center for Chemical and Biological Sciences,University of Karachi,Karachi,Sindh 75270,Pakistan;Institute of Chinese Medicine Resources,Hunan Academy of Chinese Medicine,Changsha,Hunan 410013,China;Resgreen Biological Engineering Group Co.,Ltd.,Changsha,Hunan 410329,China)
基金
Key Research and Development Project of Department of Science and Technology of Hunan Province(2022GK4015)
Natural Science Foundation of Hunan Province(2021JJ30562)
Postgraduate Research Innovation Project of Hunan University of Chinese Medicine(2021CX62)。
关键词
D-半乳糖
黄精多糖
代谢组学
肾衰老
超高效液相色谱
质谱法
D-galactose
Polygonatum sibiricum polysaccharides
Metabolomics
Kidney aging
Ultra-performance liquid chromatography(UPLC)
Mass spectrometry
